Chromosome-Specific Targeting of Dosage Compensation in Drosophila

Chromosomes are not intertwined like spaghetti, but instead occupy individual territories in the nucleus.  Sex chromosome dosage compensation in Drosophila provides a model for understanding chromosome organization and function.   Male Drosophila increase the transcript levels of genes on the single male X approximately two-fold to equal the gene expression in females, which have two X-chromosomes. Dosage compensation is mediated by the Male-Specific Lethal (MSL) histone acetyltransferase complex, which specifically binds to active genes on the male X chromosome.  Five core components of the MSL complex were identified by genetic screens for genes that are specifically required for male viability, and dispensible for females.  However, because dosage compensation must interface with the general transcriptional machinery, it is likely that identifying additional factors that are not strictly male-specific will be key to understanding the process at a mechanistic level. Here we describe two approaches to identify additional regulators.  First, we performed a cell culture-based, genome-wide RNAi screen to search for factors that facilitate MSL targeting or function.  Second, we applied a biochemical approach, ChIP-mass spectrometry (ChIP-MS), to identify MSL protein interactors on the chromatin template. Notably, we discovered a previously unstudied zinc-finger protein, CLAMP, in both screens.  CLAMP facilitates MSL targeting and displays a striking enrichment at MSL entry sites, suggesting that it may directly interact with the MSL recognition element in the first step of targeting. Our results demonstrate that the combination of crosslinking, affinity-purification, and mass spectrometry is a promising avenue for discovery of functional protein interactions associated with chromosomes in higher organisms.